PERSOONIA
Published by the Rijksherbarium, Leiden
Volume Part 8, 3, pp. 227-247 (1975)
Ultrastructure of the ascospore wall
in Pezizales (Ascomycetes) —III.
Otideaceae and Pezizaceae
Emily Merkus
Rijksherbarium, Leiden
(With Plates 34-45)
The of wall and ornamentation of is studied development layers ascospores with the electron microscope in members of the Otideaceae and Pezizaceae.
and the in Ascodesmis Primary wall, endospore, epispore developin sameway as
and the Pyronemataceae; the development of the secondary wall and the
formation of the patterns ofornamentation resemble that in the Pyronemata-
attention is ceae. Special paid to specialized plasmic structures.
Introduction
earlier I of In papers (Merkus, 1973, 1974) reported my electron microscopy the ultrastructure ofthe wall in Pezizales and discussed theresults Ascodesmis ascospore on and the Pyronemataceae sensu Eckblad (1968).
The continuationof this and the results in present paper represents a study gives
the Otideaceae and which identical species belonging to Pezizaceae, are practically with the Otideae and Aleurieae ofthe Aleuriaceae of Le Gal (1947: 284).
REVIEW OF EARLIER WORK
brief review is Like in my paper on the Pyronemataceae (Merkus, 1974), a given of Gal's of ornamentation of the of Le light microscopy the patterns ascospores the
Pezizaceae (1947). Though this involves duplication of some of the data, it gives a better overall insight and makes it easier to interpret new results. According to the rules of the international code of botanicalnomenclature some of the names used by
Le Gal (see footnotes) had to be changed.
ornamentation. The species of Pezizaceae studied by Le Gal develop simple spore
In all the wall arises and it is the species a primary ornamentation on present; orna- mentation consists of callose and pectine formations and is of sporal origin.
Persoonia, Vol. 8, Part 2 was issued 12 Sept. 1975
227 PERSOONIA Vol. Part 228 8, 3, 1975
The primary wall is covered by an "assise sous-perisporique" and a "pellicule membranaire". The "assise sous-perisporique" is formed before ornamentation
develops. The "pellicule membranaire" is termed "coque interperisporique" ifit is formed at the same time as the ornamentation and is penetrated by the substance of the the and the substance of the ornamentation; "coque interperisporique" orna- mentation both grow into one, the "coque interperisporique" also consisting of callose and pectine. The "pellicule membranaire" is termed "tunique externe de
1'assise" ifit is formed before ornamentation develops and is not penetrated by the substance of the ornamentation.
Pulparia persoonii (Crouan) Korf & al. apud Korf 1 develops simple ornamentation that is formed between the primary wall and its covering layers.
2 P. badia Pers. 3 P. P. 4 P. Peziza succosa Berk., , per Mérat, . eckinospora Karst., trachycarpa
5 6 7 Curr., P. apiculata Cooke, and P. reperta (Boud.) Moser develop simple ornamen-
which of the tation, the substance of penetrates the covering layers primary wall and is the the of the deposited on "coque interperisporique". During development ornamentation is the outside of the In the a "perispore" present on ascospores. first four species the "perispore" disappears in a later stage; in the other two species it remains.
Le Gal does of the ornamentation ofthe not give a description patterns ascospores of the Otideaceae.
of attention Though the ascoplasm is not the main subject thepresent study special is paid to it. It reveals the development of globular structures, like oil bodies and other specialized globules of a still unknown substance; these were also described by
Guilliermond (1904, 1910, 1920), the latter as "corpuscules metachromatiques".
The development of the structures and their relation to ascospore development is discussed.
MATERIALS AND METHODS
collected in the The material of the species in the present study was Netherlands,
and in the list data about the in France, Germany; following gives some specimens and their localities: Otidea alutacea (Pers. per S. F. Gray) Mass. - van Brummelen
needles of "foret de 73-501, on Picea, Liciat", Oyonnax, Ain, France, 7.X.1973
— soil The (L); Piepenbroek 837, on under Quercus, t Joppe, Gorssel, Gelderland,
O. Boud. — Brummelen the Netherlands, 12.X.1974 (L) ; bufonia (Pers.) van 4073, on ground under Betula, Schoorl, North Holland, The Netherlands, 3.IX. 1973 (L); —
1 Marcelleina Brumm. Plicaria persoonii (Crouan) Boud.; syn. persoonii (Crouan)
2 Galactinia succosa (Berk.) Sacc.
badia 2 Galactinia (Pers. per Merat) Boud. 4 Aleuria umbrina (Boud. apud Cooke) Gill.; not Peziza umbrina Pers. 2 Plicaria trachycarpa (Curr.) Boud. 6 Aleuria apiculata (Cooke) Boud.
7 Aleuria reperta (Boud.) Boud. MERKUS: Ultrastructure wall in and of ascospore Otideaceae Pezizaceae 229
under 't Piepenbroek 802, on soil, among grasses Quercus, Joppe, Gorssel, Gelderland,
The O. onotica S. F. Fckl. — Netherlands, 28.VII.1974 (L) ; (Pers. per Gray) van
the Brummelen 4638, on ground under oaks, Koningshof, Overveen, Bloemendaal,
North The 2.XI. Dur. Lév. Holland, Netherlands, 1974 (L) ; Peziza ammophila &
apud Dur. — collected during a field trip of the Dutch Mycological Society, sandy
The X. dunes, Hollumerduinen, Ameland, Friesland, Netherlands, 27. 1973 (L) ;
Pers. Pers. — Brummelen dead culms of P. ampliata per van 4074, on Phragmites,
Nederhorst den Berg, North Holland, The Netherlands, 12.V.1973 (L); P. badia
Pers. per Merat — Piepenbroek 806, on sandy soil, near estate "Dörth", Gorssel,
— Gelderland, The Netherlands, 28.VII.1974 (L) ; Piepenbroek 811, on sandy soil,
estate "Ampsen", Lochern, Gelderland, The Netherlands, 10.VIII.1974 (L); P.
Dennis — badiofusca (Boud.) Piepenbroek 746, on clay soil, Duursche Waarden between
The P. emileia Cooke — Olst and Wijhe, Overijsel, Netherlands, 23.VI. 1974 (L) ;
Piepenbroek 556, on burnt soil, "de Bannink", Colmschate, Overijsel, The Netherlands,
Dennis — 3.VI. 1973 (L); P. michelii (Boud.) van Brummelen 4500, on sandy soil,
Waterdijk, Diepenveen, Overijsel, The Netherlands, 15.VII. 1974 (L) ; P. petersii
Berk. — Siteur, on burnt soil, Eindhoven, North Brabant, The Netherlands, 21.VII.
Nannf. in Lundell — 1974 (L); P. plebeia (Le Gal) & Nannfeldt Piepenbroek 773, on
The VII. sandy soil, Epserbos, Epse, Gelderland, Netherlands, 21. 1974 (L); P.
Bres. — Brummelen burnt 't praetervisa van 4072, on soil, Joppe, Gorssel, Gelderland,
The Netherlands, 6.VIII.1973 (L); — Piepenbroek 734, on burnt soil, "Klein Noor-
The dijk", Wilp, Voorst, Gelderland, Netherlands, 27.VII.1974 (L); P. succosa
Berk. — Brummelen soil under Waarden between van 431 /, on Salix, Hengforder
Olst and The P. succosella Le Gal & Wijhe, Overijsel, Netherlands, 15.VII.1974 (L) ;
— Brummelen & soil Romagn. van Piepenbroek 4510, on under Salix, Waterdijk,
The P. Curr. — Diepenveen, Overijsel, Netherlands, 15.VII. 1974 (L) ; trachycarpa
Piepenbroek 807, on soil among mosses, near estate "Dörth", Gorssel, Gelderland, The
Netherlands, 28.VII. 1974 (L) ; P. vesiculosa Bull, per St-Am. — van Brummelen 4080,
on soil in hot-house, Kortenhoef, North Holland, The Netherlands, 28.1.1974 (L);
Pulparia persoonii Korf & al. apud Korf — Piepenbroek 575b, on loamy soil, Duursche
Waarden near Fortmond, Overijsel, The Netherlands, 19.VIII. 1973 (L) ; — Piepen- broek 738, on loamy soil, Duursche Waarden between Olst and Wijhe, Overijsel,
The Netherlands, 23.VI.1974 (L); van Brummelen 4313, on loamy soil, Duursche
Waarden between Olst and Wijhe, Overijsel, The Netherlands, 15.VII. 1974 (L);
Pustularia Fckl. — cupularis (L. per Fr.) Piepenbroek 772, on sandy soil, Epserbos, Epse,
Gelderland, The Netherlands, 21.VII. 1974 (L); Sowerbyella radiculata (Sow. per Fr.)
Nannf. — Haas, under Picea, Kreis Heidenheim, Schwabisch Alb, Germany,
— from X. 4.X. 1973 (L); exposition, Oyonnax, Ain, France, 22. 1973 (L).
After collection fromtheir substratum in the field, the apothecia were placed in the fixative. Further treatment of the apothecia followed much the same procedure as described in former studies but a few additional remarks are called for.
In addition to the former used, KMn0 or percentages 1% 4 1% glutaraldehyde was applied as primary fixative before postfixation with OSO4 to improve results. PERSOONIA Part 230 Vol. 8, 3, 1975
In order to solve the problem of inadequate impregnation of the embedding medium in the relatively hard material, in some cases the usual Epon embedding medium according to Luft ( 196 1) was replaced by a low-viscosity embedding medium according to Spurr (1961), with the additive dibutyl phtalate (Clemen$on, 1973).
The material was transferred to the Spurr embedding medium via an ethanol series,
and mixtures of the 1 00% ethanol, 100% acetone, acetone and Spurr embedding medium; polymerization lasted 12 hours at 60 °C.
The of the used of components Spurr embedding medium were at a rate 10.0 g
26.0 ERL-4206, 6.0 g D.E.R. 736, g nonenylsuccinic anhydride, 0.4 g dimethyla- minoethanoland 0.8 g dibutyl phtalate.
All sections were cut with a diamond knife.
OBSERVATIONS
The species studied so far all showed practically the same ultrastructure and a general description could be given.
ofthe data reveals thatthe ultrastructure of ofthe Otidea- A review present species ceae and Pezizaceae answers to this description. This holds particularly for the
of the delimitationof in the younger stages development; ascospores ascoplasm by unit the their distri- two delimiting membranes, form and structure of organels and bution in epiplasm and sporoplasm, and the development of the characteristic appearance of the epiplasm and sporoplasm accord completely.
of ultra- The occurrence globular structures in the ascoplasm distinguishes the
of number of structures a species. Though specific details of these globular structures will be given farther on for each species separately, some general observations can be made about them first. After use of the -fixative all ofthem are glutaraldehyde-Os0 4
and look alike. After use of the -fixative electron-transparent permanganate-Os0 4
be ranked oil bodies that less and they can as are more or electron-transparent
else other of still unknown homogeneous; or as specialized globules a substance, which are electron-dense or more moderately electron-dense and have a slightly granular structure (often with an unfixed center). In future this description is followed.
The globular structures with moderateelectron density mostly occur withboth other
and it is that intermediate types not impossible they represent forms; they are not found frequently.
It is not impossible that all three types ofglobular structures are formed in a very
of for number of it has been early stage ascus development; at any rate a species found that before meiosis and mitosis take the sites of their they are present place; the of first formationthen seem to be zones ascoplasm above and beneath the nucleus.
formation continues further Their probably during the stages of ascus development since they abound particularly after meiosis and mitosis, during the delimitationof the ascospores; in that case the zones of ascoplasm between the nuclei are also in- volved in their formation.After delimitation found in both the spore they are young and the ascospores remaining epiplasm. MERKUS Ultrastructure wall in Otideaceae and : of ascospore Pezizaceae 231
in Though some species the organization of the ascoplasmic zones above and
beneath the nucleus before meiosis and mitosis is somewhat different from that of the
zones between the nuclei after meiosis and mitosis, all of them are essentially similar
in all this in structure species; changes during ascus development in the same way.
The and the globular structures ascoplasmic zones in which they arise largely
in the the time have also in the predominate ascoplasm and, by spores developed, epiplasm.
In a the also forms of the good many species ascoplasm a large amount same
granular or more flocky material as was found in the Pyronemataceae. There it was
of supposed that both types material are related; the granular material was compared
with the glycogen particles in Ascodesmis microscopica and A. nigricans. The results of
the to confirm which has led the ofthe present study seem this, to use term glycogen the for both the granular and more flocky material. In very young stages of develop-
ment glycogen is found scattered in the ascoplasm. When the asci develop further and
the arise it abounds in clusters inside the and also ascospores just ascoplasmalemma
appears in large quantities aroundthe spores; even more than in the Pyronemataceae,
it form the of the may finally ground mass epiplasm. Moreover it may form a large
beneath the and fill the basal of the plug just ascus top, may part ascus completely.
It is also present in small quantities in the sporoplasm.
of fixative determine Per- The types applied the appearance of the glycogen.
seems to it and it manganate-0s0 4 preserve adequately gives a fairly electron-dense,
or more structure. 0s0 granular flocky Glutaraldehyde-Os0 4 (or 4 only; Merkus,
overall makes the 1 973) gives poorer results, as was also found by Schrantz (1968) ; it
less electron-dense and it the glycogen (to electron-transparent) seems to change
at location of the glycogen, sometimes not fixing it at all or else concentrating it a
few places, thereby causing large vacuoles in the epiplasm.
The of the walls is in Ascodesmis and the development spore much the same as
Pyronemataceae; between the two delimiting unit membranes wall material suc-
cessively forming the primary and secondary walls is deposited; at first the primary
wall is in it to homogeneous appearance but in a later stage of development seems
differentiate into an outer epispore and an inner endospore; the secondary wall arises between the wall is primary and the outer delimiting membrane that now
called investing membrane; the inner delimiting membrane becomes the sporo-
plasmalemma. Primary wall, epispore, and endospore show exactly the same de-
velopment and ultrastructure as those in Ascodesmis and the Pyronemataceae. For a
ultra- general description compare my earlier studies. The development and the
structure of the secondary wall have much in common with those in the Pyrone-
mataceae; they will be described in detailfor each species separately.
As found Ascodesmis loses was in and the Pyronemataceae the epiplasm gradually
the from the formationof vacuoles organels originally present; apart glycogen, large
that often have flocky contents develop in the epiplasm of many species. When the
thin spores mature the epiplasm disintegrates almost completely; only a layer of
inside the remain. In the all original plasm just ascoplasmalemma may sporoplasm PERSOONIA- Vol. Part 232 8, 3, 1975
from increase in electron its total organels remain present; apart a general density
in the of oil appearance changes many species through development large drops.
OTIDEACEAE
OTIDEA ALUTACEA, O. BUFONIA, AND O. ONOTICA
Fixatives: and Of the three permanganate-Os0 4 glutaraldehyde-Os0 4 . species
Otidea bufonia showed the best fixation; the description of the younger stages of
development applies only to this species; the mature spores at any rate do not differ.
asci before meiosis and mitosis show normal without Young a ascoplasm any clusters of been formed. special unusual structures; glycogen have already In the
found in same stage however changes are the endoplasmic reticulum; at some places
it widens slightly, becomes electron-transparent internally and may form circular
structures (PI. 34A); it may also widen furtherand form small vacuoles with vesicles
sizes local increase in electron be found of varying (PI.34B); finally a density may
(PI. 34A, B); a relation with the electron dense globules, evidently arising in this
is not Whetherthere is connection between these stage, impossible (PI. 34A, B). any
changes and the presence of glycogen is not clear.
of show further structural in Following stages development changes the ascoplasm
and formation of the spores. The epiplasm forms increasing amounts of glycogen,
and vacuoles with fairly electron-dense flocky contents; the organels disappear.
The sporoplasm increases in electron density and develops oil drops. The primary
wall (permanganate-0s0 glutaraldehyde-Os0 nm thick), the 4 350-450 nm, 4 250
nm and the nm, epispore (25 thick), endospore (permanganate-Os0 4 250 glutaral-
200 nm are normal in from the normal dehyde-0s0 4 thick) appearance; apart
striation in the epispore the endospore shows some internal differentiation(PI. 34C,
D, G). The development of the secondary wall proceeds regularly; it consists of
homogeneous or more flocky material and varies from 100-1000 nm in thickness;
inclusions from the sometimes epiplasm are present (PI. 34C, D).
The -fixed material could not be observed in older permanganate-Os0 4 stages.
In all three species it has been found in the glutaraldehyde-Os0 -fixed material that 4 it part of the contents of the secondary wall concentrates on the epispore. Here
forms smooth that each have thickness of about two succeeding layers a 70 nm;
though connected, they remain clearly distinguishable (PI. 34F, G). The structures
that have been found in the secondary wall of Otidea onotica and, to some extent,
of also in that of O. alutacea possibly represent transitional forms in the development
the two layers (PI. 34E).
By the time the spores have matured the epiplasm and the rest of the secondary
wall have disappeared; the condensed layers on the epispore seem to remain. MERKUS: Ultrastructure wall in Olideaceae and of ascospore Pezizaceae 233
PUSTULARIA CUPULARIS
Fixatives: and permanganate-0s0 4 glutaraldehyde-0s0 4.
The show a wall 100-200 youngest stages present primary (permanganate-Os0 4
nm that has a thin inner of an obvi- nm, glutaraldehyde-Os0 4 50-100 thick) layer
loose structure and outer which in the slides ously a homogeneous layer appears as
circular band the of the an uninterrupted, along spores. Separation investing mem-
occurred the whole intermediate which brane has along primary wall. The space,
varies in thickness from 100-1500 nm, has been filled up with fairly electron-dense
flocky material forming the secondary wall; it also contains inclusions like vesicular
structures that must have been derived from the epiplasm or from the investing
membrane, which in both fixatives runs irregularly. Epiplasm and sporoplasm are
normal in appearance (PI. 35A).
this of the it From stage development on, epiplasm disintegrates slowly; appears
loose and flocky, like the contents of the secondary wall; the organels in it disappear.
be increases Little glycogen seems to formed. At the same time the sporoplasm
in electron and oil In the - greatly density develops drops. glutaraldehyde-Os0 4
fixed material some concentration of secondary wall material is found near the
distributed in primary wall or the secondary wall (PI. 35B).
In and the is succeeding stages an epispore an endospore evolve; epispore 30 nm
thick and has normal in the striation; as Sepultaria, endospore (permanganate-0s0 4
nm show a broad electron- 150-250 nm, glutaraldehyde-Os0 4 100-150 thick) may
dense and thin and sometimes electron-d' outer part a interrupted fairly nse layer
in the innermore parts (PI. 35C, D).
In the mature asci the epiplasm has dissolved completely and almost disappeared,
leaving only a small zone at the innerside of the ascus wall. The secondary wall has also been broken down; the remnants of the investing membraneremain the longest.
The mature spores are smooth (PI. 35D).
SOWERBYELLA RADICULATA
Fixative: from the available glutaraldehyde-Os0 4 . Though pictures nothing
can be said about the development of the ascoplasm and, in later stages, of the
few still be made about the epiplasm, a remarks can spores.
The first of show that the of the wall stages development structures primary
not (250-400 nm thick) and the sporoplasm do differ from the general description;
the sporoplasm develops some oil drops. In following stages an epispore (25 nm
thick) and an endospore (150—200 nm thick) have differentiated and the secondary
wall has been formed between the investing membraneand the primary wall.
The wall material has electron-dense it is secondary a granular structure, though
sometimes found be further the to more homogeneous. During spore development
secondary wall material concentrates on the primary wall, where ornamentation
the wall internal striation of the elements crops up. Perpendicular to primary an PERSOONIA Vol. Part 234 8, 3, 1975
ofornamentationis evident in the before their electron has in- younger stages density
creased that far This with fibrous that have been (PI. 35E). may agree structures found in the secondary wall (PL 35F).
The mature spores have tapering or more rounded spines about 50-250 nm high,
the ends ofthe The intervals and at spores up to 700 nm high. spines occur at regular
are connected by a smooth layer ofabout 1 o nm thick; growing together is also to be
found (PI. 35G).
PEZIZACEAE
PEZIZA AMMOPHILA AND P. PRAETERVISA
Fixatives: and permanganate-Os0 4 glutaraldehyde-Os0 4.
In these the above and beneath the in two species ascoplasmic zones nucleus,
which the less in globular structures start to arise, are more or partitioned an early
of the central is subdivided into kind of membrane stage development; ascoplasm a
enveloped plasmic compartments between which glycogen is found, and the more superficial ascoplasm contains much glycogen gathered in clusters (PI. 36A).
The in the elements of the organels compartments are: endoplasmic reticulum, which appear as tubular or vesicular structures; larger vesicles that usually show
electron and be derived and poor density may from the endoplasmic reticulum;
membranous structures which are globular and seem to consist of a moderately
electron-dense center that might be glycogen and that is wrapped up in a varying
number in the of membranes, possibly invaginating the center. At many places
membranes the diffuse in The vesicles enveloping compartments are appearance. with electron also found in the poor density are superficial ascoplasm.
The globular structures, which are electron-dense in these species, are particularly evident of the in the glycogen the superficial ascoplasm but are also found between
in the central compartments ascoplasm (PI. 36A).
In later stages of development, in which meiosis and mitosis take place and spore
delimitation and into starts (PI. 37A), the compartments enlarge considerably grow
vacuoles with basic substance the large a flocky (PI. 36B). Simultaneously ascoplasm
between the nuclei becomes essentially similar in appearance, whereby the glycogen and the electron-dense globular structures are conspicuous. Large vacuoles however are not found here; theelements of the endoplasmic reticulum and the membranous structures cluster in small vacuoles (PI. 36C).
Once the formed the ofthe in the spores are development plasm proceeds epiplasm.
Here the amount of glycogen proceeds to increase; the glycogen also fills the basal
of the part ascus completely.
Both the primary wall and the sporoplasm are regular in appearance. In Peziza
the wall nm, glutaraldehyde- praetervisa primary (permanganate-0s0 4 400-500
0s0 nm is but sometimes shows a thin and 4 200-300 thick) electron-transparent fairly electron-dense intermediate layer. In Peziza ammophila the primary wall MERKUS in and : Ultrastructure of ascospore wall Otideaceae Pezizaceae 235
(permanganate-0s0 400-600 glutaraldehyde-0s0 11m thick) is 4 nm, 4 300-500 completely homogeneous (PI. 37C). In the sporoplasm both the electron-dense
globular structures and an abundant endoplasmic reticulumare present; the electron
density has increased (PI. 37B, C, D).
Separation of the investing membrane occurs along the whole primary wall,
in some places becoming fairly conspicuous; the investing membrane runs straight.
The secondary wall that is formed between the primary wall and the investing
membrane has fairly electron-dense and homogeneous contents (PI. 37C, D).
the much that In Peziza praetervisa amount of glycogen increases in this stage so
only a small strip of epiplasmic structures remains between the glycogen and the
secondary wall. In Peziza ammophila large vacuoles with flocky electron-dense
around the but for the these vacuoles contents develop spores; most part disappear
again and are replaced by large clusters of glycogen. Once the secondary wall has
reached its ultimate thickness in and in (locally 200 nm Peziza ammophila 500 nm
P. praetervisa) all organels in the epiplasm will have disappeared; the epiplasm then
consists only of glycogen, in which the electron-dense globular structures are no
longer conspicuous; they shrivel and finally disappear. In the sporoplasm two oil
drops develop; the electron-dense globular structures remain.
During the development of the secondary wall an epispore and an endospore
the P. evolve; epispore (Peziza praetervisa: 35-40 nm thick; ammophila: 35-50 nm
shows normal thick) a pattern of differentiation; the endospore (Peziza praetervisa:
nm P. permanganate-Os0 4 300-400 nm, glutaraldehyde-Os0 4 150-250 thick;
ammophila: permanganate-Os0 glutaraldehyde-Os0 nm 4 500-1500 nm, 4 350-500 sometimes has thin in the innermost thick) a layer with increased electron density
part (PI. 37B, D, E, F). When the the spores mature the contents of the secondary wall concentrate on
in both in smooth epispore species (PI. 37D, E). In Peziza ammophila this results a
electron-dense about in which differentiation be layer 70 nm thick, internal can
distinguished; it shows subtle striation that is surrounded by a heavier layer (PI.
In smooth 37F). Peziza praetervisa ornamentation is formed consisting of a fairly
electron-dense about intervals small layer 40 nm thick, at regular punctuated by
rounded warts the elements of ornamentation do approximately 300 nm high; not
show internal ofthe any structure (PI. 37B). In both species the epiplasm and the rest secondary wall disappear.
PEZIZA VESICULOSA
Fixatives: permanganate-Os0 and glutaraldehyde-Os0 . 4 4 The of the appearance ascoplasm of this species agrees fairly well with that of the
the similar in two preceding species; structures are but not so abundant as Peziza
ammophila and P. praetervisa; the ascoplasm above and beneath the nucleus shows subdivision into compartments in the initial stages of development, particularly in
the ofthe In this both basal upper part ascus (PI. 38A, B). species the part and some PERSOONIA Vol. Part 236 8, 3, 1975
of the superficial part of the ascoplasm consist of glycogen in this stage, the basal
part clearly containing the remnants of the original ascoplasm.
into becomes After delimitationof the spores, subdivision plasmic compartments
more evident in the epiplasm beneath the lowermost spore. The membranes forming
the compartments may show numerous invaginations; they may also separate
locally and have larger areas with glycogen; in the glycogen the globular structures
are found (PI. 38C). In contrast to those in Peziza ammophila and P. praetervisa these
develop as fairly electron-transparent structures with somewhat flocky contents
in The (PI. 38B) and become only a later stage more electron-dense (PI. 38C).
between the does epiplasm spores develops an essentially corresponding structure, as that in Peziza ammophila and P. praetervisa.
In following a primary wall (permanganate-0s0 nm thick) is stages 4 500-900
formed (PI. 39A, B) which seems to differentiate into an epispore (35-50 nm thick)
and an nm endospore (permanganate-0s0 4 500-2000 thick) (PI. 39C, D, E, F);
the secondary wall arises between the primary wall and the investing membrane,
its local The of the thickness amounting to about 700 nm (PI. 39B, C). appearance
wall this also of the wall accords with those in primary and, at stage, secondary
Peziza ammophila and P. praetervisa. The structures ofthe epiplasm and the sporoplasm
vacuoli- are also similar to those in P. ammophila and P. praetervisa; in the epiplasm
zation and formation of large clusters of glycogen are found; in the sporoplasm no
oil drops are formed.
The last stages of development of the spores are the same as in Peziza ammophila.
The concentrationof the secondary wallmaterial on the epispore (PI. 39C, D) results
in a rather smooth electron-dense layer (about 100 nm thick), which shows a similar
the internal structure, subtle striation surrounded by a heavier layer (PI. 39E, F);
epiplasm and the rest of the secondary wall disappear.
PEZIZA P. P. P. MICHELII, PLEBEIA, SUCCOSA, AND SUCCOSELLA
four have Fixatives: and . These species permanganate-Os0 4 glutaraldehyde-Os0 4
much in be described so common that they can together.
Young asci before meiosis and mitosis show a rather vague partition of the as-
coplasm above and beneath the nucleus into a central part and a more superficial
electron-dense endo- part, the latter consisting mainly of normal ascoplasm; poorly
vesicles and scattered both The of the plasmic glycogen are over parts. appearance
central is the that of the internuclear in Peziza part practically same as ascoplasm
ammophila and P. praetervisa;; the elements of the endoplasmic reticulum together with
numerous membranous structures cluster in small vacuoles. In Peziza succosa and
P. succosella the membranous structures are globular; in P. michelii and P. plebeia
reniformor dumb-bell-shaped structures are also present (Pis. 40A, B, C; 41A, B, C).
Characteristic four is to ofthe species thatthe numerous globular structures prove
be less which all the more or electron-transparent oil bodies, are present over as-
coplasm. Though not always clearly visible in this young stage of development the MERKUS: wall in Otideaceae and Ultrastructure of ascospore Pezizaceae 237
oil bodies embedded in the in later when the of are glycogen; stages, amount glycogen
increases, this becomes more evident (Pis. 40C; 41B, C). Though young asci were
difficult to find it appeared that in Peziza michelii and in P. plebeia all the structures
in asci that have been formed are present just (PI. 41 A).
After delimitation of the spores the epiplasm between the nuclei will have devel-
oped similar structures, and the amount of both the oil bodies and the glycogen will
have increased. The is found in clusters all the largely glycogen over ascus, particu-
larly in the apical and basal part; in the latterit has completely replaced the epiplasm.
The sporoplasm contains numerous oil bodies. In Peziza michelii and P. plebeia the
does further in in P. epiplasm not change a succeeding stage of development; succosa
and P. succosella real vacuoles may arise.
The develop normally. The primary wall spores (permanganate-0s0 4 150-350
nm, 100-200 nm does not show internal differ- glutaraldehyde-Os0 4 thick) any
entiation before the endospore (permanganate-Os0 4 150-250 nm, glutaraldehyde-
0s0 100-200 nm and the michelii and P. nm 4 thick) epispore (Peziza plebeia: 45-55
P. and P. succosella: evolve. is thick; succosa : 50-60 nm thick) The endospore practically
homogeneous in appearance; the epispore shows the ordinary layered structure
The membraneand the (Pis. 42; 43). secondary wall is formed between the investing
wall. It has electron-dense in primary flocky, fairly contents and may vary widely
thickness; the investing membrane runs almost straight (Pis. 42A, B; 43A). In this
of wall stage development the epiplasm slowly disintegrates; just inside the ascus
it remains the oil bodies orientated longest. Here the become elongated and perpen-
to dicular the ascus wall; they sometimes develop a somewhat flocky content. In-
vaginations from the epiplasm into the secondary wall have been found with both
offixative The oil and types (Pl. 43A, C). sporoplasm develops large drops a some-
times conspicuous endoplasmic reticulum.
Before the differentiationof the primary wall the contents of the secondary wall
into electron-dense continuous concentrate globules, flattened globules or more
the wall In the layers on primary (Pis. 42B; 43C, F). following stages secondary wall material gradually concentrates, and warts and ridges connected by a smooth
thinlayer arise on the primary wall at regular intervals. During further maturationof
the both spores the epiplasm and the rest of the secondary wall disappear.
In and first show Peziza succosa P. succosella, the elements of ornamentation at
internal differentiation. In the -fixed material the basal permanganate-0s0 4 parts of elements the are fairly electron-dense and maintain a rather loose structure, the
condensed the material that borders the epispore showing subtle striation all over
the electron-dense and and spore surface; upper parts are fairly homogeneous seem
to have been added as a kind of In the glutaral- separately cap (Pis. 42C, D; 43B).
-fixed material the loose structure ofthe basal show striation dehyde-Os0 4 parts may
to the the and be perpendicular spore surface; upper parts are electron-dense may covered by an electron-transparent layer (PI. 42F, G).
In later ornamentation stages the internal differences in the elements of finally
in the -fixed materialand the ornamentation becomes disappear permanganate-0s0 4 PERSOONIA Vol. Part 238 8, 3, 1975
electron-dense (PI. 42E). In glutaraldehyde-Os0 -fixed material of Peziza succosella 4
the material remains and to be surrounded electron-transparent present seems by an
electron-dense layer (PI. 42H).
In Peziza succosa and P. succosella ornamentation is rather similar. In P. succosa warts 600-800 nm high, which are sometimes slightly broadened or may have
into found with smaller grown ridges, are together warts about 150-300 nm high;
in P. succosella and connected smooth about thick warts ridges are by a layer 50 nm (PI. 42E, H).
In michelii Peziza and P. plebeia the concentrationofsecondary wall material on the
have increased since be epispore seems to more regularly separate parts cannot easily
less solid remain and form distinguished; only parts seem to permanent gaps (PI.
In P. ornamentation in the in P. 43D, E). plebeia develops exactly same way as
-fixed material could not be studied in the latest michelii; glutaraldehyde-Os0 4
of The ornamentationconsists ofrounded which in stages development. warts, vary size from 200-700 nm and may have broadened or grown together; the connective layer varies in thickness from 60-100 nm.
PEZIZA BADIA
Fixative: permanganate-Os0 4.
this resembles Though species closely Peziza michelii, P. plebeia, P. succosa, and
P. succosella in all of it has in with stages development, a particular amount common the first two mentioned. of differences species In an early stage ascus development are found in the fact that the membranous structures are almost never globular, but
in somewhat reniform, dumb-bell-shaped or other ways irregular (PI. 44A). In a later clusters of take the of the in the stage large glycogen place organels epiplasm.
The of the wall the aspects primary (300 nm thick), epispore (45-55 nm thick),
does and the endospore (150-200 nm thick) agree with the general description, as
of condensation the aspect the secondary wall. Like in Peziza michelii and P. plebeia the of wall material the different in the devel- secondary proceeds fairly regularly; parts oping elements of ornamentation, clearly present in P. succosa and P. succosella, are not formed.
The ornamentation is surrounded by an electron-dense layer that is evidently formed its it consists of and by outermost part; warts ridges (100-400 nm high), which may form an incomplete network and which are connected by a smooth layer about thick 40 nm (PI. 44B).
PEZIZA BADIOFUSCA, P. EMILEIA, AND P. PETERSII
Fixative: As an formation is in the permanganate-Os0 4 . incipient spore present youngest stages studied, it could not be concluded what kind of development the ascoplasm undergoes. MERKUS: wall in Otideaceae and Ultrastructure of ascospore Pezizaceae 239
The structures of the epispore (Peziza badiofusca and P. petersii: 60 nm thick;
P. emileia: 40-50 nm thick) and the endospore (P. badiofusca: 200-250 nm thick;
do differ from P. emileia: 500-800 nm thick; P. petersii: 250-35° nm thick) not the
general description (Pis. 44C, D, E, F; 45A, B). The endospore sometimes has a thin electron-dense layer in the innermore parts; in P. petersii several electron-dense
be layers may present (PI. 44F). The secondary wall has also developed; it varies largely in thickness along the
surface of a single spore; its contents are fairly electron-dense and homogeneous,
membrane almost sometimes slightly flocky (PI. 44C). The investing runs straight.
The epiplasmic organels have nearly all disappeared and have been replaced by
glycogen; the remainder is found close to the ascus wall. Here they may occur
together with the same electron-dense globular structures as those found in other
species, which at this place may be elongated and orientated perpendicular to the
ascus wall. The sporoplasm has an increased electron density and has developed oil
drops; it often shows a rather abundant endoplasmic reticulum.
The of ornamentation resembles that in development spore preceding species. redistribution wall internal In the first stages of of the secondary material differences
divide the elements of ornamentationin various In the basal parts. Peziza badiofusca
of the elements are electron-dense and the parts homogeneous, intermediate parts electron-dense the have a fairly and loose structure, and upper parts are fairly electron-denseand homogeneous (PI. 45A). In P. emileiaand P. petersii the developing elements of ornamentationare homogeneous in structure, the upper parts being less electron-dense than the lower parts, in which in P. petersii subtle striation is sometimes
In in P. the internal distinguishable (PI. 44C, D, E, F). some places petersii structure
ofthe elements is which In later all loose, causes permanent "gaps" (PI. 44E). stages
the internal differences disappear and the elements of ornamentationbecome com-
pletely electron-dense in all three species (PI. 45B).
In the mature of Peziza ornamentation is formed spores badiofusca by rounded, isolated and the surface warts, 300-700 nm high spread regularly on spore ; they are
connected by a rather smooth layer about 50 nm thick in which small warts are
sometimesfound (Pl. 45B). In P. emileiaornamentationis formed by slender, isolated
which connected smooth warts (500-800 nm high), are by a layer (about 30 nm
thick) and occur at regular intervals (PI. 44D). In P. petersii warts and ridges (300-
500 nm high) are also found at regular intervals along the spore surface and are
connected smooth in the oldest of by a layer (60-70 nm thick); stages development
the of the further and the sometimes tops warts may enlarge grow out laterally, warts fusing (PI. 44F).
Like in Peziza badia,, ornamentation in P. badiofusca and particularly that in P.
petersii is surrounded by a marked electron-dense layer (Pis. 44E, F; 45B). In all
three species the epiplasm and therest of the secondary wall have disappeared when
the spores are mature. PERSOONIA Vol. Part 240 8, 3, 1975
PEZIZA TRACHYCARPA
Fixative: In the discernible the permanganate-0s0 4 . youngeststages development
of the primary wall is complete and a secondary wall has already started to form.
Therefore, and as a result of relatively poor preservation, no conclusions could be
about the first of the drawn stages ascoplasm.
The wall is 600-800 thick and has normal primary nm a aspect; separation of the
investing membrane from the primary wall has made formation of the secondary
wall possible; this has homogeneous and fairly electron-dense contents. In the
remnants of the epiplasm glycogen is found and the same oil bodies occur as in
several other species. The sporoplasm has fairly electron-dense contents with an
extensive endoplasmic reticulum; it has developed one large oil drop, sometimes
accompanied by smaller ones.
In of normal and a following stage development a epispore (60 nm thick) endospore
differentiate (200-250 nm thick) and, though in most places the investing membrane
has gone, it can be seen that locally the secondary wall increases further in thickness.
The wall becomes in which be primary extremely wavy outline, must seen as an
artefact.
time the At the same secondary wall material concentrates on the epispore.
emileia and first Like in Peziza P. petersii, at the basal parts ofthe developing elements of ornamentation electron-dense and while the are homogeneous, upper parts are
fairly electron-dense and homogeneous (PI. 45C). A locally loose structure of the elements also found may cause permanent "gaps", as was in P. petersii. In later stages
the elements of ornamentation become completely electron-dense. Together with
the epiplasm, the rest of the secondary wall material disappears.
When the spores mature the upper parts of the elements of ornamentationgrow
but out laterally, no longer forming warts, umbrella-shaped structures (1200-1500
nm high) that sometimes fuse at the edges. In between the epispore is covered by a
smooth (50 nm thick). their is worth layer Finally, temporary "hairy" appearance this is in mentioning; not found any of the other species studied (PI. 45D).
PULPARIA PERSOONII
Fixative: Preservation of this to be permanganate-Os0 4 . species appears difficult; this made a complete study of it impossible.
As regards the contents of the ascoplasm and their distribution over the ascus,
show close to Peziza P. P. P. and young stages similarity badia, michelii, plebeia, succosa,
the first three P. succosella; particularly species agree in that the membranous structures are globular or more irregularly formed. A noteworthy difference with these species is the absence of oil bodies and the presence of electron-dense globular structures (often with an unfixed center, as sometimes found in other species) resembling those in Peziza ammophila, P. praetervisa, and others. MERKUS: Ultrastructure wall in and ofascospore Otideaceae Pezizaceae 241
The eventual development of vacuoles in the epiplasm is uncertain; glycogen is
present in large quantities and takes the place of most ofthe original epiplasm. In the
oil sporoplasm large drops develop. The development of the spore walls is normal;
the epispore (50 nm thick) and the endospore (250-350 nm thick) seem to differenti-
ate fromthe primary wall (300-400 nm thick); the secondary wall develops between the primary wall and the separating investing membrane.
At first the secondary wall material is homogeneous and fairly electron-dense.
its redistribution from When starts, electron-dense spots arise at regular distances
each other the surface. As the of on spore they gradually enlarge, remaining contents
the secondary wall seem to be restructured into fibrous elements; these easily join and
give the developing elements of ornamentation a freakish appearance (PL 45E).
It is not clear whether these structures result from normal development or are caused
by incomplete fixation. In the mature spores ornamentation is formed by irregular
warts (400-800 nm high) that maintain a fibrous outer layer (PI. 45F).
DISCUSSION
On the results of this electron confirm those of earlier many points microscopy
studies. Not only does the general ultrastructure of each species agree with what is
of but — already known Ascomycetes the way in which the ascospores start to develop
delimitationof the in the unit membranes and spores ascoplasm by two delimiting
the formation of spore walls between these two delimiting unit membranes — bears
out similar developments in Ascodesmis (Merkus, 1973), in the Pyronemataceae
(Merkus, 1974), and in other Ascomycetes (Reeves, 1967; Carroll, 1966, 1967,
1969; Delay, 1966; Wells, 1972; Schrantz, 1966, 1970).
Later of reveal structural resemblance both stages development a strong to
Ascodesmis and the The different of Pyronemataceae. appearance two successively
formed spore wall layers has again led to the use of the terms primary and secondary
and the involved in walls; processes the formation of these spore walls, the differ- entiation of the primary wall into the epispore and the endospore, the internal
differentiationof the epispore and the endospore, the various reactions of the spore
wall layers on the fixatives applied, together with the structural changes of the
and also it will suffice refer epiplasm sporoplasm, agree. Perhaps, therefore, to to
discussions previous on these subjects.
A marked difference with the results obtained thus far is the of the appearance
young ascoplasm of the genera Otidea, Peziza, and Pulparia. Globular structures,
like oil bodies and other specialized globules, are present in most of the species;
the more or less electron-transparent oil bodies are restricted to Peziza ; the other
specialized globules, which are electron-dense or more moderately electron-dense,
exist in Otidea and and found in number of of Peziza. prove to Pulparia are a species
The could be studied this The genus Sowerbyella not adequately on point. genus Pustularia is more usual in its internal structure and lacks the globular structures.
It is difficult decide to what processes are involved in the development of the PERSOONIA Vol. Part 242 8, 3, 1975
which often found embedded in It is im- globular structures, are glycogen. not
that the membranous structures in the central possible ascoplasm may play a role, though interventionofthe poorly electron-densevesicles derived from the endoplasmic
reticulum must also not be overlooked. More knowledge about their chemistry
might reveal something more about these processes. In this connection the develop-
of of Otidea ment ofthe ascoplasm species seems to represent a simple form ofthose in
the other species.
structures food this also assumed The globular are probably a reserve; was by
Guilliermond (1904, 1910, 1920). They do not seem to play an active role in the
formation of the spore walls. In the epiplasm they slowly lose their contents and
the absorbed the disappear when spores mature, not by being by spores (Guillier-
mond) but by shrinkage or shriveling. In the sporoplasm they seem to persist; the
oil oil bodies join to large drops, like in Peziza badia, P. michelii, P. plebeia, P. succosa,
and P. succosella; the other specialized globules maintain their original form. When
absent in the the still oil in later oil bodies are ascoplasm spores may develop drops
like Otidea O. O. P. stages, in alutacea, bufonia, onotica, Peziza ammophila, badiofusca,
P. emileia, P. petersii, P. praetervisa, P. trachycarpa, Pulparia persoonii, and Pustularia
vesiculosa oil all. cupularis. In Peziza no drops are formed at
A food reserve is also provided in large quantities by glycogen. Though the possi-
bility of chemical changes or the influence of the fixative cannot be precluded it
structural forms of exist from the seems as ifother glycogen apart typical glycogen
particles that have been described (a.o. by Schrantz, 1968, for Peziza plebeia).
The of the wall and the structural it aspect secondary changes undergoes during
the development of ornamentation is the same as in the Pyronemataceae. The
secondary wall material is at first regularly spread in the secondary wall and is
similar in appearance to the older epiplasm in most of the species. It seems to be
redistributed in later stages and condenses or concentrates on the epispore, where it
forms ornamentation.Like in the the involved a complete Pyronemataceae, processes
in this condensation or concentration of secondary wall material are regular and
characteristic for each species separately. A continuous addition of new secondary
wall materialduring the formationof ornamentationis not impossible. The fateofthe
remaining secondary wall materialand the investing membrane is unknown, though
it is highly likely that it disappears with the epiplasm.
Like in the Pyronemataceae this development of ornamentationis found in all the
species with ornamented spores, viz. Peziza badia, P. badiofusca, P. emileia, P. michelii,
P. petersii, P. plebeia, P. praetervisa, P. succosa, P. succosella, Pulparia persoonii, and
Sowerbyella radiculata. Apart from the striation in the developing ornamentation of
Sowerbyella radiculata, the thin surrounding layer that is present in Peziza badia, P.
ornamentation badiofusca, and P. petersii, and the somewhat fibrous outer part of the of Pulparia persoonii, no further internal differentiationin the ornamentationis found
in of the any species.
In comparing the development of the smooth spores in the Pyronemataceae and
in ofthis it similarities in Pustularia the species study appears that exist; cupularis some Ultrastructure wall Otideaceae and Pezizaceae MERKUS : of ascospore in 243
condensation ofsecondary wall material is found but it disappears in a later stage so
forms ofthe In Peziza that ultimately the epispore the outermost part mature spores.
and P. vesiculosa however condensation of wall ammophila a permanent secondary
material causes the formation of extra, smooth layers on the epispore. A similar
O. and addition of smooth layers on the epispore occurs in Otidea alutacea, bufonia,
0. onotica it is uncertain whether these but layers are permanent.
Though the terminology of the spore wall layers is somewhat different my results
Pustularia and Peziza with those of Schrantz on the species cupularis plebeia agree
(1966, 1970). His descriptions for both the development oftwo succeeding spore wall
layers, the "couche primaire" (primary wall) and the "perispore" (secondary
wall) in both species, and the formation of the "couche ornementale" in the "peri-
of similar those in the He also found differen- spore" Peziza plebeia are to present study. inner tiation of the "couche primaire" into the outer "exospore" (epispore) and the "epispore" (endospore) in both species, and further development of the innermost
and "endospore" (not described as a particular layer by me) in Pustularia cupularis;
and "masses mentioned the presence of an "ectospore" (investing membrane)
denses" (plasmic inclusions).
As the hand similarities to exist between the ultrastructureof on one so many prove
other the species of the Pyronemataceae, Otideaceae, and Pezizaceae and, on the
Gal's observations these also I add further details hand, Le on taxa agree, can no
to the comparison of the two studies. To avoid further duplication I refer to previous
discussions on the subject.
ACKNOWLEDGEMENTS
The author is greatly indebted to Professor Dr. C. Kalkman, Director of the
their Rijksherbarium (Leiden), and to Dr. J. van Brummelen for stimulating and
discussions. thanks also due Miss B. A. D. Dam and Mrs. F. P. critical Many are to
Boers-van der Sluijs for their skilled technical assistance, to Mrs. G. Piepenbroek-
Groters for sending valuable living materialof many species, and to Mrs. E. Helmer
van Maanen Ph. D. for suggestions about the manuscript. This study could not have
been carried out without the financial support of the Foundationfor Fundamental
Biological Research (BION), which is subsidized by the Netherlands Organization
for Advancement of Pure Research (ZWO).
REFERENCES
CARROLL, G. C. (1966). A study of the fine structure of ascosporogenesis in Saccobolus kervemi
and Ascodesmis sphaerospora. Ph. D. Thesis. University of Texas, Austin [unpublished].
The of delimitation in In (1967). ultrastructure ascospore Saccobolus kerverni. J. Cell
Biol. 33: 218-224.
(1969). A study of the fine structure of ascosporogenesis in Saccobolus kerverni. In Arch.
Mikrobiol. 66: 321-339.
CLEMENgoN, H. (1973). Die Wandstrukturen der Basidiosporen. III. Cortinarius und Der-
In Z. Pilzk. mocybe. 39: 121-144.
243 PERSOONIA Part 244 Vol. 8, 3, 1975
DELAY, C. (1966). Etude de l'infrastructure de l'asque d’Ascobolus immersus Pers. pendant la
maturation In Annls Sci. XII des spores. nat. (Bot.) 7: 361-420.
The of the A re-evaluation of their ECKBLAD, F.-E. (1968). genera operculate Discomycetes.
and nomenclature. In Bot. taxonomy, phylogeny Nytt Mag. 15: 1-191.
A. de la formation des GUILLIERMOND, (1904). Contribution a asques et de 1'epiplasme des Ascomycetes. In Revue g6n. Bot. 16: 49-66.
A de volutine. In Arch. (1910). propos des corpuscules metachromatiques ou grains
Protistenk. 19: 289-309. (1920). Sur les relations entre le chondriome des champignons et la m<5tachromatine.
In C.r. Seanc. Soc. Biol. 83: 855-858. Recherches les ornementations des LE GAL, M. (1947). sur sporales Discomycetes operculfe.
In Annls Sci. XI 8: nat. (Bot.) 73-297.
H. in resin methods. In biochem. LUFT, J. (1961). Improvements epoxy embedding J. biophys.
Cytol. 9: 409-414.
E. of wall in Pezizales MERKUS, (1973). Ultrastructure the ascospore (Ascomycetes)—I.
Ascodesmis and A. In Persoonia microscopica (Crouan) Seaver nigricans van Tiegh. 7: 351-366. Ultrastructure of the wall in Pezizales (1974). ascospore (Ascomycetes)—II. Pyrone-
mataceae sensu Eckblad. In Persoonia 8: 1-22.
The fine of formation in domesticum. REEVES, JR., F. (1967). structure ascospore Pyronema In
Mycologia 59: 1018-1033. de de chez SCHRANTZ, J.-P. (1966). Contribution a l'<5tude la formation la paroi sporale
Fuck. In Paris 26a: Pustularia cupularis (L.) C. r. hebd. Séanc. Acad. Sei., 1212-1215. (1968). Ultrastructure et localisation du glycogène chez l'Ascomycète Galactinia plebeia
Le Gal. In Rev. Cytol. Biol. v6g. 31: 151-154.
(1970). Etude cytologique, enmicroscopie optique et électronique, de quelquesAscomy-
cètes. II. La In Rev. Biol. m-168. paroi. Cytol. v6g. 33:
A resin medium for electron SPURR, A. R. (1969). low-viscosity epoxy embedding microscopy.
In Res. 26: J. Ultrastruct. 31-43. WELLS, K. (1972). Light and electron microscopic studies of Ascobolus stercorarius. II. Ascus
In Pubis. Bot. and ascospore ontogeny. Univ. Calif. 62: 1-93.
EXPLANATION PLATES OF 34-45
ABBREVIATIONS USED IN PLATES. — AW, ascus wall; CM, condensed material; E, epiplasm;
En, endospore; Ep, epispore; ER, endoplasmic reticulum; EV, endoplasmic vesicle; G, glycogen; GS, globular structure; IAM, inner ascospore-delimiting membrane; IM, investing membrane; MS, membranous structure; OAM, outer ascospore-delimiting membrane;
N, nucleus; PI, plasmic inclusions; PW, primary wall; S, sporoplasm; SW, secondary wall;
T, tonoplast; V, vesicle; Va, vacuole.
PLATE 34
Figs. A-D. Otidea bufonia, fixed in 1.5% KMnC>4 and 1% OsO,» and stained with uranyl
and lead citrate: before acetate Figs. A, B. ascoplasm, spore development, X 18,200; Fig. C.
of D. id. also spore development, development secondary wall, X 29,000; Fig. showing develop- ment of epispore and endospore, X 29,000.
E. Otidea of Fig. onotica, spore development, temporary internal structure condensing secondary wall material, fixed in 1% glutaraldehyde and 1% OsC>4 and stained with uranyl
and lead acetate citrate, X 29,700. Merkus: infrastructure of ascospore wall in Otideaceae and Pezizaceae 245
F. Otidea condensation of wall Fig. alutacea, spore development, secondary material, fixed
in i% glutaraldehyde and i% OsC>4 and stained with uranyl acetate and lead citrate, X 18,500.
Fig. G. Otidea bufonia, detail of condensed secondary wall material, fixed in 1 % glutaralde-
stained with lead hyde and 1% OSO4 and uranyl acetate and citrate, X 115,500.
PLATE 35
A-D. Pustularia stained with lead citrate: A. Figs. cupularis, spore development, Fig. begin-
of wall fixed in and B. ning secondary formation, 1% KMnC>4 1% OSO4, X 29,700; Fig.
further development of secondary wall, with condensation of secondary wall material, fixed in
1% glutaraldehydeand 1% 0s0 X Fig. C. development of and endospore, 4, 29,700; epispore in D. fixed and 0s0 4 x id. 1% glutaraldehyde 1% , 36,300; Fig. E-G. fixed in and Figs. Sowerbyella radiculata, spore development, 3.25% glutaraldehyde
1% OSO4: Fig. E. condensation ofsecondary wall material and development of epispore and endospore, stained with uranyl acetate and lead citrate, X 36,300; Fig. F. id. showing fibrous
structure of secondary wall material, stained with lead citrate; Fig. G. advanced state in
and lead development of ornamentation, stained with uranyl acetate citrate, X 29,700.
PLATE 36
Figs. A-C. Peziza praetervisa, ascoplasm, fixed in 1.5% KMnO, and 1% OSO4 and stained
with acetate and lead before meiosis and uranyl citrate: Fig. A. upper part of ascus mitosis,
B. of after meiosis and of X 16,600; Fig. upper part ascus mitosis, X 8,400; Fig. C. ascoplasm
the between the same ascus, nuclei, X 8,400.
PLATE 37
B. in and Figs. A, Peziza praetervisa, spore development, fixed 1.5% KMnC>4 and 1% OsC>4 stained with and lead citrate: A. of uranyl acetate Fig. young stage spore development, just
after B. advanced in the of spore delimitation, x 14,900; Fig. state development ornamenta-
tion, epispore, and endospore, X 18,200.
C-F. fixed in and Figs. Peziza ammophila, spore development, 1.5% KMnC>4 and 1% OsC>4
stained with uranyl acetate and lead citrate: Fig. C. beginning of secondary wall formation, X 18,200; Fig. D. condensation of secondary wall material and development of epispore and
E. F. of endospore, x 9,900; Fig. id. X 18,200; Fig. id. detail, advanced state spore devel- opment, X 36,300.
PLATE 38
A-C. Peziza fixed in KMnCAt and A. Figs. vesiculosa, ascoplasm, 1.5% 1% OSO4: Fig. upper
of before meiosis and stained with and part ascus mitosis, uranyl acetate lead citrate, X 9,900; stained with Fig. B. basal part of ascus before meiosis and mitosis, uranyl acetate and lead
basal of delimitation of with lead citrate, X 17,300; Fig. C. part ascus after spores, stained
citrate, X 17,300.
PLATE 39
A-F. in and Figs. Peziza vesiculosa, spore development, fixed 1.5% KMn04 1% OSO4:
A. formation of stained with and Fig. young ascospore during primary wall, uranyl acetate lead citrate, X 18,200; Fig. B. beginning of secondary wall formation, stained with uranyl PERSOONIA Part 246 Vol. 8, 3, 1975
and lead C. condensation of wall material acetate citrate, X 9,900; Fig. secondary and devel- opment of epispore and endospore, stained with lead citrate, X 18,200; Fig. D. id.; Fig. E. F. of stained id.; Fig. id. advanced state spore development, with uranyl acetate and lead
citrate, X 21,400.
PLATE 40
B. KMn0 and and with Figs. A, Peziza succosa, ascoplasm, fixed in 1% 4 1% 0s04 stained
and lead citrate: A. of before meiosis and uranyl acetate Fig. upper part ascus mitosis, X 7, 100 ; Fig. B. detail, X 16,600. 1
Fig. C. Peziza succosella, id. X 5,800.
PLATE 41
A. of before and in Fig. Peziza michelii, ascoplasm, upper part ascus meiosis mitosis, fixed
KMn0 and 0s0 and stained with acetate and lead 1% 4 1% 4 uranyl citrate, X 7,400.
Figs. B, C. Peziza plebeia, id.: Fig. B. X 7,100; Fig. C. detail, X 21,400.
PLATE 42
Figs. A-H. Peziza succosella, spore development: Fig. A. beginning of secondary wall forma-
in 0s0 with acetate tion, fixed 1 % KMn04 and 1 % 4 and stained uranyl and lead citrate,
X 29,700; Fig. B. id. condensation ofsecondary wall material and developmentof epispore and endospore, stained with lead citrate; Fig. C. id. stained with uranyl acetate and lead citrate;
D. lead of Fig. id. stained with citrate, X 33,300; Fig. E. id. advanced state spore development, stained with and lead of wall uranyl acetate citrate, X 29,700; Fig. F. condensation secondary material and development of epispore and endospore, fixed in 1% glutaraldehyde and 1%
0s0 and stained with acetate and lead X G. H. id. 4 uranyl citrate, 29,700; Fig. id.; Fig.
of advanced state spore development.
PLATE 43
B. fixed in and and Figs. A, Peziza succosa, spore development, 1% KMn04 1% 0s0 4 stained and lead A. of wall with uranyl acetate citrate, X 29,700 : Fig. beginning secondary formation and some condensation ofsecondary wall material; Fig. B. condensation ofsecond-
wall and of and ary material development epispore endospore.
and Figs. C-E. Peziza michelii, spore development, stained with uranyl acetate lead citrate,
C. condensation of wall fixed in X 29,700: Fig. secondary material, 1% glutaraldehyde and
0s0 D. id. also of and fixed in 1% 4 ; Fig. showing development epispore endospore, 1% 0s0 KMn04 and 1% 4 ; Fig. E. id.
F. condensation of wall fixed in Fig. Peziza plebeia, spore development, secondary material,
and 0s0 and stained with acetate and lead X 1 % glutaraldehyde 1 % 4 uranyl citrate, 29,700.
PLATE 44
B. fixed in KMn0 and 0s0 and stained with Figs. A, Peziza badia, 1 % 4 1 % 4 uranyl acetate and lead A. detail of in of before meiosis citrate, X 23,100: Fig. ascoplasm upper part ascus and mitosis; Fig. B. condensation of secondary wall material and development of epispore and of endospore, advanced state spore development. MERKUS: Ultrastructure of ascospore wall in Otideaceae and Pezizaceae 247
D. fixed in and Figs. C, Peziza emileia, spore development, i% KMnC>4 and i% OsC>4
with lead of stained uranyl acetate and citrate, X 29,700: Fig. C. condensation secondary wall material and development of epispore and endospore; Fig. D. id. advanced state of
spore development.
F. fixed in Figs. E, Peziza petersii, spore development, 1% KMnC>4 and 1% OsC>4 and stained with uranyl acetate and lead citrate: Fig. E. condensation of secondary wall material of id. advanced of and development epispore and endospore, X 29,700; Fig. F. state spore
development, X 33,300.
PLATE 45
B. fixed in and and Figs. A, Peziza badiofusca, spore development, 1% KMn04 1% OSO4 stained with uranyl acetate and lead citrate: Fig. A. condensation of secondary wall material
and of B. id. of development epispore and endospore, X 29,700; Fig. advanced state spore development, X 23,100.
D. fixed in and Figs. C, Peziza trachycarpa, spore development, 1 % KMnC>4 and 1 % OSO4 stained with uranyl acetate and lead citrate: Fig. C. condensation of secondary wall material and of and D. id. advanced of development epispore endospore, X 29,700; Fig. state spore development, X 23,100.
F. Figs. E, Pulparia persoonii, spore development, stained with uranyl acetate and lead citrate: Fig. E. condensation of secondary wall material and development of epispore and
fixed in and F. id. advanced of endospore, 1.5% KMn04 1% OsC>4, X 29,700; Fig. state
in and spore development, fixed 1 % KMn04 1 % OSO4, X 36,300. PLATE PERSOONIA 8(3) (MERKUS) 34 PERSOONIA 8(3) (MERKUS) PLATE 35 PLATE 36 PERSOONIA 8(3) (MERKUS) PERSOONIA 8(3) (MERKUS) PLATE 37 PERSOONIA 8(3) (MERKUS) PLATE 38 PLATE PERSOONIA 8(3) (MERKUS) 39 PLATE PERSOONIA 8(3) (MERKUS) 40 PERSOONIA PLATE 8(3) (MERKUS) 41 PERSOONIA PLATE 8(3) (MERKUS) 42 PLATE PERSOONIA 8(3) (MERKUS) 43 PERSOONIA 8(3) (MERKUS) PLATE 44 PERSOONIA PLATE 8(3) (MERKUS) 45